Recombinant DNA technology has made it possible to express genes of one organism within another. The prior art shows that several virus groups, including the papovaviruses, papiluoma viruses, adenoviruses and retroviruses have been employed as eukaryotic molecular cloning and expression vectors. The relatively small sizes of these virus genomes have facilitated the in vitro construction of recombinant DNA molecules. However, these vectors generally exhibit a limited host range, provide severe limitations on the amounts of DNA that can be accommodated and lose their ability to infect subsequent cells upon the insertion of foreign DNA. Genetic engineering using larger viruses, i.e. those having genomes larger than about 50 kbp, such as poxviruses, herpesviruses or baculoviruses, is more difficult because of the large genome size. However, unlike viruses with smaller virus genomes, the larger virus genomes have a greater capacity for accommodating large foreign nucleic acid sequences. Poxviruses are particularly useful because the viruses can infect a wide range of host cells. For poxviruses, such as vaccinia virus, past methods for generating recombinants have employed homologous recombination.
Prior to the present invention, homologous recombination in vivo was used to introduce foreign DNA into the genomes of large DNA viruses such as herpesviruses, poxviruses, and baculoviruses (reviewed by Miller, Bioessays 11: 91-5 (1989); Moss, Science 252: 1662-7 (1991) and Roizman, et al., Science 229: 1208-14 (1985)). Homologous recombination requires a number of genetic manipulations no longer required by the methods disclosed herein. As a first step for practicing homologous recombination, a vector is prepared separate from the viral genome. The vector is preferably a plasmid and in a poxvirus system, the plasmid is modified to contain a poxvirus promoter, sites for insertion of a foreign gene, and poxvirus DNA flanking sequences. A foreign gene is next inserted into the vector to form a chimeric gene and this construct is transfected into cells infected with a poxvirus having sequences complementary to the DNA flanking sequences. The progeny poxviruses are collected and tested for the presence of the foreign gene.
Direct in vitro ligation of DNA, into large eukaryotic viral genomes has not been used. It has heretofore been thought that such ligation techniques are not possible because of the difficulty of working with vector genomes of 50 to 200 kilobase pairs (kbp). With longer DNA molecules there is an increase in the number of times that a particular restriction endonuclease recognition site appears in the molecule and this creates significant problems for vectors created using methods other than homologous recombination. There has been a long felt belief in the scientific community that large genomes of this size are incapable of efficient direct ligation. For example, European Patent Application No. 0 443 335 to Bodemer indicates that the size of the vaccinia genome makes the construction of recombinant genomes by cleavage with restriction endonucleases and subsequent ligation with foreign DNA impossible. Accordingly, homologous recombination methods have been used to insert foreign DNA into viruses having greater than 50 kbp.
There are, however, significant problems associated with homologous recombination. For example, the overall efficiency of homologous recombination is low and the efficiency continues to decline further with increasing insert length. Moreover, the exact site of incorporation of a foreign DNA insert can only be predicted within a given region of the vector genome. The exact site of incorporation varies for each individual recombination event within that given region. For this reason, considerable effort has gone into the development of selection and screening methods. Another potential drawback of homologous recombination protocols is that they generally require an intermediate cloning step followed by propagation of the DNA in bacteria where deletions or rearrangements may occur. Deletions and rearrangements are even more prevalent when the DNA has an unusual structure or is very large. These intermediate plasmid cloning steps make the production of cDNA expression libraries extremely labor intensive.
Recombinant viruses with large genomes, such as vaccinia virus, that have been generated by homologous recombination, have been shown to be useful as vaccines to generate protective immune response against the organisms from which the foreign DNA of the chimeric gene was derived. Some examples of such foreign genes include nucleic acid sequences encoding protein from hepatitis B virus, hepatitis A virus, hepatitis non-A, non-B virus, influenza virus, herpesvirus, cytomegalovirus, adenoviruses, parvoviruses, foot and mouth disease virus, poliovirus, measles virus, rabies virus, coronaviruses, coxsackieviruses and pathogenic bacteria, rickettsia, protozoa, and metazoa.